Remodeling of the postsynaptic proteome in male mice and marmosets during synapse development

Postsynaptic proteins play crucial roles in synaptic function and plasticity. During brain development, alterations in synaptic number, shape, and stability occur, known as synapse maturation. However, the postsynaptic protein composition changes during development are not fully understood. Here, we show the trajectory of the postsynaptic proteome in developing male mice and common marmosets. Proteomic analysis of mice at 2, 3, 6, and 12 weeks of age shows that proteins involved in synaptogenesis are differentially expressed during this period. Analysis of published transcriptome datasets shows that the changes in postsynaptic protein composition in the mouse brain after 2 weeks of age correlate with gene expression changes. Proteomic analysis of marmosets at 0, 2, 3, 6, and 24 months of age show that the changes in the marmoset brain can be categorized into two parts: the first 2 months and after that. The changes observed in the first 2 months are similar to those in the mouse brain between 2 and 12 weeks of age. The changes observed in marmoset after 2 months old include differential expression of synaptogenesis-related molecules, which hardly overlap with that in mice. Our results provide a comprehensive proteomic resource that underlies developmental synapse maturation in rodents and primates.

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Toru Takumi Jan 18, 2024
The MS MS and MS/MS data were searched against the NCBI-nr using Proteome Discoverer version 2.2 (Thermo Fisher Scientific) with the MASCOT search engine software version 2.6 (Matrix Science).
Statistical analyses were performed using R software (version 3.4.4and 4.1.0).Heatmaps were generated in in using the heatmap.2function in in the gplots package.For k-means clustering, the number of of clusters was assessed using the NbClust package.One-way analysis of of variance (ANOVA) was performed using the oneway.testfunction.Correction of of p-value with Benjamini-Hochberg method was performed using the p.adjust function.The correlation coefficient was calculated using the cor.test function.Fisher's exact test was performed using the fisher.testfunction.The detailed R codes used in in this study are available on on GitHub (https://github.com/Takeshi-Kaizuka/Kaizuka_Proteomics_2024).Enrichment of of proteins reported to to be be expressed on on PSD was evaluated using DAVID version 6.8.GO GO analysis and pathway analysis was performed using Metascape and SynGO.Enrichment of of transcription factor binding on on the genes was analyzed using ChIP-Atlas.Enrichment of of disease-related genes and transcription binding sites were analyzed using ToppCluster.Canonical pathway analysis and network analysis were performed using Ingenuity Pathway Analysis (QIAGEN).other socially relevant groupings

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We didn't perform sample-size calculation.As for mouse samples, we chose n=4 to examine statistical significance of the results, considering the maximum number of samples that we can manipulate at once.As for marmoset samples, we chose n=2 doe to the limitation of sample availability In the proteome analyses, we extracted major proteins according to the three criteria and eliminated the other proteins from the following analysis unless otherwise stated; (1) at least two unique peptides were identified, (2) quantified in all datasets, and (3) coefficient of variation < 100.In the case when multiple proteins are encoded by a single gene, we selected a single protein encoded by a single gene whose signal intensity is highest.
We performed the experiments taking biological replicates as described in the manuscript to confirm the reproducibility of the results.There is no more successful or unsuccessful replication data that are not described in the manuscript.
No randomization was performed in this study.Considering the technical variation of sample preparation, we grouped the subsets of individual age (e.g. 2, 3, 6, and 12-week-old) and repeated preparation of indicated number of samples.
In this study, we didn't apply blinding of the samples for following reasons.
(1) Because different number of mouse brains was needed to prepare single PSD sample from different age (see Methods), we couldn't apply blinding.
(2) There is some differences in appearance of marmoset brain at different age, which makes the blinding difficult.
The sample preparation and mass spectrometry was performed by different researchers and the latter researcher didn't have any biological hypothesis or bias.So, the lack of blinding doesn't affect the results of this study.

April 2023
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